Abstract Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNPs (U1, U2, U4, U5, U6) and many non-snRNP related protein factors. The spliceosome undergoes dramatic changes in a splicing cycle, generating the E, A, Pre-B, B, Bact, B*, C, C*, P, and ILS complexes. High resolution cryoEM structures of A, pre-B, B, Bact, C, C*, P, and ILS complexes have been determined in the last few years, generating intriguing hypotheses on the mechanism of splicing that await rigorous testing through biochemical and/or genetic analyses. The cryoEM structure of the post-catalytic P complex we determined at 3.3 resolution provided intriguing hypothesis on the importance of intronic secondary structures in 3? ss recognition. In this proposal, we will use genome-wide chemical and enzymatic probing to test this hypothesis. The structure of the P complex also led to hypotheses on the structure and function of DEAD-box helicase Prp22 in splicing, which we will test using a combination of biochemical and genetic experiments. Furthermore, the cryoEM structure of the spliceosome E complex we just determined suggested mechanism of exon definition and its conversion to intron definition, which we will test using biochemical and structural approaches. The combination of genomic, biochemical, and structural approaches used in this proposal has the potential to provide a complete mechanistic understanding of the entire splicing cycle.